Download NucleoSpin® 96 Plant II (Core Kit) - MACHEREY

Genomic DNA
from plant
User manual
NucleoSpin® 96 Plant II
NucleoSpin® 96 Plant II Core Kit
May 2014 / Rev. 03
Genomic DNA from plant
Table of contents
1 Components 4
1.1 Kit contents
4
1.2 Reagents to be supplied by user
5
2 Product description
6
2.1 The basic principle
6
2.2 Kit specifications
6
2.3 Required hardware
7
2.4 Recommended accessories for use of the NucleoSpin® 96 Plant II Core Kit 8
2.5 Automated processing on robotic platforms
9
2.6 Storage and homogenization of samples
10
2.7 Elution procedures
11
3 Storage conditions and preparation of working solutions
12
4 Safety instructions
13
5Protocols
15
5.1NucleoSpin  96 Plant II – centrifuge processing
15
5.2NucleoSpin® 96 Plant II – vacuum processing
20
®
6Appendix
26
6.1Troubleshooting
26
6.2 Ordering information
27
6.3 Product use restriction / warranty
29
MACHEREY-NAGEL – 05 / 2014, Rev. 03
3
Genomic DNA from plant
1
Components
1.1 Kit contents
NucleoSpin® 96 Plant II
2 x 96 preps
4 x 96 preps
24 x 96 preps
740663.2
740663.4
740663.241
Lysis Buffer PL1
125 mL
250 mL
6 x 250 mL
Lysis Buffer PL22
100 mL
200 mL
6 x 200 mL
Precipitation Buffer PL3
25 mL
50 mL
6 x 50 mL
Binding Buffer PC
125 mL
250 mL
6 x 250 mL
Wash Buffer PW1
100 mL
2 x 100 mL
12 x 100 mL
Wash Buffer PW2
(Concentrate)2
100 mL
2 x 100 mL
12 x 100 mL
Elution Buffer PE3
60 mL
125 mL
6 x 125 mL
RNase A (lyophilized)2
30 mg
2 x 30 mg
12 x 30 mg
NucleoSpin® Plant II
Binding Plate
(dark green rings)
2
4
24
MN Wash Plate
2
4
24
Rack of Tube Strips4
(for lysis and elution)
4
8
32
Cap Strips
24
48
288
MN Square-well Block
6
12
72
Gas-permeable Foil
10
20
120
User manual
1
1
1
REF
1
2
3
4
The kit for 24 x 96 preparations REF 740663.24 consists of 6 x REF 740663.4.
For preparation of working solutions and storage conditions see section 3.
Composition of Elution Buffer PE: 5 mM Tris/HCl, pH 8.5
1 rack = 12 strips with 8 tubes each, Cap Strips included
4
MACHEREY-NAGEL – 05 / 2014, Rev. 03
Genomic DNA from plant
1.1 Kit contents continued
NucleoSpin® 96 Plant II Core Kit
4 x 96 preps
REF
740468.4
Lysis Buffer PL1
250 mL
Lysis Buffer PL21
200 mL
Precipitation Buffer PL3
50 mL
Binding Buffer PC
250 mL
Wash Buffer PW1
2 x 100 mL
Wash Buffer PW2 (Concentrate)1
2 x 100 mL
Elution Buffer PE2
125 mL
RNase A (lyophilized)1
2 x 30 mg
NucleoSpin® Plant II Binding Plate
(dark green rings)
4
User manual
1
1.2 Reagents to be supplied by user
•
1
2
96–100 % ethanol
For preparation of working solutions and storage conditions see section 3.
Composition of Elution Buffer PE: 5 mM Tris/HCl, pH 8.5
MACHEREY-NAGEL – 05 / 2014, Rev. 03
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Genomic DNA from plant
2
Product description
2.1 The basic principle
The NucleoSpin® 96 Plant II kit is designed for the isolation of genomic DNA from plant
materials. After the plant samples have been homogenized, the DNA can be extracted
with lysis buffers containing chaotropic salts, denaturing agents and detergents. The
standard isolation ensures the lysis of plant material with the CTAB Lysis Buffer PL1,
which is specially developed for plants. In addition, an SDS based lysis buffer, Buffer
PL2, is provided as an alternative. Buffer PL2 requires subsequent protein precipitation
with potassium acetate. Lysates should be cleared by centrifugation in order to remove
polysaccharides, contaminations, and residual cellular debris. The clear supernatant
is mixed with Binding Buffer PC to create conditions for optimal binding to the silica
membrane in the binding plate. After washing with two different buffers (Buffer PW1
and Buffer PW2) DNA can be eluted in low salt Buffer PE or water and is ready-to-use
for subsequent analysis and processing.
2.2 Kit specifications
6
•
NucleoSpin® 96 Plant II is designed for the isolation of genomic DNA from
plant material.
•
NucleoSpin® 96 Plant II allows parallel purification of multiples of 96 samples
each with up to 100 mg sample per well (wet weight).
•
Depending on the individual sample, NucleoSpin® 96 Plant II shows yields in
the range of 1–30 μg DNA (maximum column capacity is about 30 μg) with an
A260/A280 ratio between 1.80 and 1.90 and typical concentrations of 100–200 ng/
μL. The amount of DNA that can be expected per mg of sample extracted
depends on the size and ploidy of the genome. For example, 100 mg fresh
wheat with a hexaploid genome (1.7 x 1010 bp) contain 30 μg DNA, whereas
the same amount of Arabidopsis with a smaller diploid genome (1.9 x 108 bp)
yields only 3 μg DNA.
•
The eluted DNA is ready-to-use in subsequent reactions like PCR, restriction
analysis, etc.
•
NucleoSpin® 96 Plant II can be processed under vacuum or in a centrifuge.
•
Two lysis buffers, based on CTAB (PL1) or SDS (PL2) are provided.
•
NucleoSpin® 96 Plant II can be used manually with the NucleoVac 96 Vacuum
Manifold (see ordering information) or other vacuum devices.
MACHEREY-NAGEL – 05 / 2014, Rev. 03
Genomic DNA from plant
Kit specifications at a glance
Parameter
NucleoSpin® 96 Plant II
Technology
Silica-membrane technology
Format
96-well plates
Processing
Manual or automated, vacuum or centrifugation
Sample material
20–100 mg plant tissue, plant cells (wet weight)
Fragment size
50 bp–approx. 50 kpb
Typical yield
1–30 μg
A260/A280
1.8–1.9
Elution volume
100–200 μL
Preparation time
60 min/plate (excl. lysis)
Binding capacity
30 μg
2.3 Required hardware
NucleoSpin® 96 Plant II can be processed under vacuum or with centrifugation.
Certain hardware for processing is required.
Centrifugation
For centrifugation, a microtiterplate centrifuge is required. This centrifuge must be able
to accomodate the NucleoSpin® Plant II Binding Plate stacked on a Round- or Squarewell Block and reach accelerations of 5,600–6,000 x g is required (bucket height:
85 mm).
Regarding waste collection, suitable consumables (e.g., MN Square-well Blocks) are
necessary and they are not included in the kit. For the most convenient handling,
without the need of emptying and reusing the MN Square-well Blocks, we recommend
using six MN Square-well Blocks if two 96-well plates are processed at once (see
ordering information). Alternatively, it is possible to empty the MN Square-well Blocks
after every centrifugation step, reducing the amount of MN Square-well Blocks needed.
Vacuum processing
The NucleoSpin® 96 Plant II kit can be used with the NucleoVac 96 Vacuum Manifold
(see ordering information). When using NucleoSpin® 96 Plant II with less than 96
samples, Self-adhering PE Foil (see ordering information) should be used in order to
MACHEREY-NAGEL – 05 / 2014, Rev. 03
7
Genomic DNA from plant
close and protect non-used wells of the NucleoSpin® Plant II Binding Plate and thus
guarantee proper vacuum.
Establish a reliable vacuum source for the NucleoVac 96 Vacuum Manifold. The
manifold may be used with a vacuum pump, house vacuum, or water aspirator. We
recommend a vacuum of -0.2 to -0.4 bar (reduction of atmospheric pressure). The
use of the NucleoVac Vacuum Regulator (see ordering information) is recommended.
Alternatively, adjust the vacuum so that during the purification the sample flows through
the column with a rate of 1–2 drops per second. Depending on the amount of sample
being used, the vacuum times may need to be increased for complete filtration.
Additionally, a suitable centrifuge for sample preparation steps may be required.
For general consumables and equipment needed, please see section 1.2.
2.4 Recommended accessories for use of the NucleoSpin®
96 Plant II Core Kit
The NucleoSpin® 96 Plant II Core Kit provides all necessary buffers, enzymes and
NucleoSpin® Binding Plates. Accessories (e.g., lysis plates, waste collection plates,
elution plates or tubes) are not provided with the Core Kit. The reduced kit composition
along with a large variety of separately available accessories, allow optimal adjustment
of the kit to individual user needs. The user can select additional consumables according
to his requirements for highest flexibility.
The NucleoSpin® 96 Plant II Core Kit provides buffers RNase A, and NucleoSpin®
Binding Plates only. Accessory plates (e.g., elution plates) are not provided with the
core kit. The user can individually select additional consumables from a variety of
suitable accessory plates according to his requirements for highest flexibility. For use of NucleoSpin® 96 Plant II Core Kit follow the standard protocols (see section
5.1 or 5.2, respectively).
Recommended accessories for use of the NucleoSpin® 96 Plant II Core Kit are available from MACHEREY-NAGEL. For ordering information please refer to section 6.2.
8
MACHEREY-NAGEL – 05 / 2014, Rev. 03
Genomic DNA from plant
Protocol step
Suitable consumables,
not supplied with the core kits
1. Homogenize
samples
Rack of Tube Strips
with Cap Strips
4. Adjust binding
conditions
Square-well Block
or
Remarks
For mixing cleared
lysate with Buffer
PC
Round-well Block
or
MN Square-well Block
7. Wash silica
membrane
MN Wash Plates
8. Elute DNA
Rack of Tubes Strips
with Cap Strips
MN Wash Plate
minimizes the
risk of cross
contamination
(vacuum
processing)
or
Round-well Block
or
Round-well Block Low
(centrifugation only)
MACHEREY-NAGEL – 05 / 2014, Rev. 03
9
Genomic DNA from plant
2.5 Automated processing on robotic platforms
NucleoSpin® 96 Plant II can be used fully automated on many common laboratory
workstations. For the availability of scripts and general considerations about adapting
NucleoSpin® 96 Plant II on a certain workstation please contact MN. Full processing
under vacuum enables complete automation without the need for centrifugation steps
for drying of the binding membrane or for elution.
The risk of cross-contamination is reduced by optimized vacuum settings during the
elution step and by the improved shape of the outlets of the NucleoSpin® Plant II
Binding Plate.
Drying of the NucleoSpin® Plant II Binding Plates under vacuum is sufficient because
the bottom of the plate is protected by the MN Wash Plate during the washing steps.
As a result, it is recommended to integrate the MN Wash Plate into the automated
procedure to protect against these wash buffer residues. The MN Frame (see ordering
information) can be used to position the disposable MN Wash Plate inside the vacuum
chamber. This also reduces the risk of cross-contamination as common metal adaptors
tend to get contaminated by gDNA. In addition, thorough cleaning of the vacuum
chamber is recommended after each run to prevent forming of gDNA-containing
aerosols.
Visit MN online at www.mn-net.com or contact your local MACHEREY-NAGEL
distributor for technical support regarding hardware, software, setup instructions, and
selection of the protocol. Several application notes of the NucleoSpin® 96 Plant II
kit on various liquid handling instruments can also be found at www.mn-net.com at
Bioanalysis / Literature.
2.6 Storage and homogenization of samples
We recommend using young plant samples and keeping the plants in the dark for
about 12 h before collecting samples (if possible) in order to reduce the polysaccharide
content.
Plant samples can be stored frozen, under ethanol, or lyophilized. In many cases
lyophilized, dried material can be processed more easily and gives higher yield.
However, keep in mind that dried samples may reduce the amount of starting material
by the factor 5 (for example, 20 mg dried plant leaves vs.100 mg fresh weight).
As plant tissue is very robust, the lysis procedure is most effective with well
homogenized, powdered samples. Suitable methods include grinding with pestle and
mortar in the presence of liquid nitrogen or using steel beads. We also recommend the
use of other commercial homogenizers, bead mills, etc.
Methods to homogenize samples
•
10
Commercial homogenizers, for example Crush Express for 96-well homogenization (Saaten-Union Resistenzlabor GmbH, D-33818 Leopoldshöhe), Tissue
Striker (www.KisanBiotech.com), or Geno/Grinder 2000 can be used.
MACHEREY-NAGEL – 05 / 2014, Rev. 03
Genomic DNA from plant
•
Samples can be disrupted using bead based homogenization tools, for example, GenoGrinder (http://www.spexcsp.com or for Germany www.c3-analysentechnik.de) or Mixer Mill MM400 (http://www.retsch.com/products/milling/ballmills/mm-400/). Please refer to instrument manufacturers recommendations for
suitable plates or tubes for homogenization.
•
Homogenizing samples by VA steel beads (diameter: 3 mm): Put 4–5 beads
and plant material together into a 15 ml plastic tube (Falcon), chill the tube
in liquid nitrogen, and vortex for about 30 seconds (e.g., with a Multi Pulse
Vortexer, Schütt Labortechnik GmbH, Postfach 3454, D-37024 Göttingen,
Germany). Repeat this chilling and vortexing procedure until the entire plant
material is ground to a powder. Chill the tube once more and remove the beads
by rolling them out gently or remove them with a magnet. Keep the material
frozen throughout the whole homogenization procedure. Do not add nitrogen to
the tube! This leads to sticking and loss of plant material attached to the beads.
•
High-throughput homogenization: Add the plant tissue to the individual tubes of
the Tube Strips. Add one 3 mm stainless steel bead to each tube and close the
individual tubes with Cap Strips. Freeze the sample in liquid nitrogen and insert
the Rack of Tube Strips in a suitable homogenization tool (e.g., mixer mill).
For disruption, shake the samples for 60–90 s at 30 Hz or until a homogenous
plant powder has been formed. If necessary, repeat shaking once. Fresh plant
material can also be homogenized with lysis buffer, however, homogenization
of fresh plant material with lysis buffer may cause shearing of DNA. For
frozen plant material thawing should be avoided during the homogenization.
Samples should be frozen in liquid nitrogen before homogenization. Lyophilized
or silica-gel dried material can be homogenized with or without lysis buffer.
Homogenization of lyophilized tissue with lysis buffer may result in higher yield
but also may cause shearing of DNA.
2.7 Elution procedures
It is possible to adjust the elution method and the volume of the elution buffer to the
specific application of interest. In addition to the standard method (recovery rate about
80–90 %) described in the protocols, there are 3 modifications possible:
•
High yields: 90–100 % of bound nucleic acids can be eluted by performing two
elution steps with volumes as indicated in the protocol, for example 2 x 100 μL.
Finally, combine eluates and measure yield.
•
Alternatively, use preheated Elution Buffer PE (70 °C): Preheat elution buffer
to increase yield. After loading half of the preheated elution buffer (50 μL) onto
the membrane, incubate the NucleoSpin® Plant II Binding Plate for 3 min at
60–70 °C. Centrifuge for elution as indicated. Repeat the elution step once.
•
Highly concentrated eluates: Using a minimal elution volume (about 50 μL)
about 70–80 % of bound nucleic acids can be eluted, resulting in highly
concentrated eluates.
MACHEREY-NAGEL – 05 / 2014, Rev. 03
11
Genomic DNA from plant
Elution may also be performed with Tris-EDTA-buffer (TE) with a pH equal or higher
than 8. This will increase DNA stability during long term or multi-use storage at 4 °C (or
ambient temperature) by inhibiting omnipresent DNases. However, EDTA interferes,
depending on the final concentration, with certain downstream applications. For optimal performance of isolated DNA in downstream applications, we recommend
eluting with the supplied elution buffer and storing it, especially long term, at -20 °C.
Several freeze-thaw cycles will not interfere with most downstream applications.
Performance of long-range PCR (e.g., > 10 kbp), or the detection limit of trace amounts
of DNA species, may be reduced after multiple freeze-thaw cycles or prolonged storage
of eluted DNA at 4 °C or room temperature. This is due to shearing of DNA or adsorption
to surfaces.
Due to the dead volume of the silica membrane please note that the difference between
the dispensed elution volume and the recovered elution buffer is approximately 45 μL
(recovered elution volume = dispensed elution volume - 45 μL).
12
MACHEREY-NAGEL – 05 / 2014, Rev. 03
Genomic DNA from plant
3
Storage conditions and preparation of working
solutions
Attention: Buffer PL1 contains CTAB, Buffer PL2 contains SDS, Buffers PC and PW1
contain chaotropic salt! Wear gloves and goggles!
CAUTION: Buffers PC and PW1 contain guanidine hydrochloride which can form highly
reactive compounds when combined with bleach (sodium hypochlorite). DO NOT add
bleach or acidic solutions directly to the sample-preparation waste.
•
Store RNase A at 4 °C on arrival (storage at 4 °C may cause precipitation of
salts in different buffers).
•
All other components can be stored at room temperature (18–25 °C) and are
stable for up to 1 year.
Before starting any NucleoSpin® 96 Plant II protocol prepare the following:
•
Lysis Buffer PL2: Check for precipitated SDS especially after storage at
temperatures below 20 °C. If necessary incubate the bottle for several minutes
at 30–40 °C and mix well until the precipitate is redissolved completely.
•
Wash Buffer PW2: Add the indicated volume of ethanol (96–100 %) to Buffer
PW2 Concentrate before first use. Store Buffer PW2 at room temperature (18–
25 °C) for up to one year.
•
RNase A: Add the given volume of water (indicated on the vial, see below) to
lyophilized RNase A. Store the RNase A solution at 4 °C for up to 3 months. For
longer storage (up to 1 year), the RNase A solution should be divided into small
aliquots and stored at -20 °C.
NucleoSpin®
96 Plant II
NucleoSpin®
96 Plant II
NucleoSpin®
96 Plant II
NucleoSpin®
96 Plant II
Core Kit
2 x 96 preps
4 x 96 preps
24 x 96 preps
4 x 96 preps
740663.2
740663.4
740663.24
740468.4
Wash
Buffer PW2
(Concentrate)
100 mL
Add 400 mL
ethanol
2 x 100 mL
Add 400 mL
ethanol to
each bottle
12 x 100 mL
Add 400 mL
ethanol to
each bottle
2 x 100 mL
Add 400 mL
ethanol to
each bottle
RNase A
(lyophilized)
30 mg
Add 2.5 mL
H2O
2 x 30 mg
Add 2.5 mL
H2O
to each vial
12 x 30 mg
Add 2.5 mL
H2O
to each vial
2 x 30 mg
Add 2.5 mL
H2O
to each vial
REF
MACHEREY-NAGEL – 05 / 2014, Rev. 03
13
Genomic DNA from plant
4
Safety instructions
The following components of the NucleoSpin® 96 Plant II and NucleoSpin® 96 Plant II
Core kits contain hazardous contents.
Wear gloves and goggles and follow the safety instructions given in this section.
GHS classification
Only harmful features need not be labeled with H and P phrases until 125 mL or 125 g.
Mindergefährliche Eigenschaften müssen bis 125 mL oder 125 g nicht mit H- und P-Sätzen gekennzeichnet
werden.
Component Hazard contents
GHS symbol
Hazard Precaution
phrases phrases
Inhalt
Gefahrstoff
GHS Symbol
H-Sätze
P-Sätze
PC
Guanidinium hydrochloride 24–36 % + ethanol
35–55 %
226, 302
210, 233,
301+312, 330,
403+235
226, 302,
319
210, 233, 280,
301+312,
305+351+338,
330, 337+313,
403+235
317, 334
261,
304+340,
342+311,
301+312,
280,
302+352,
333+313
PW1
RNase A
Warning
Guanidiniumhydrochlorid
24–36 % + Ethanol 35–55 %
Achtung
Guanidinium hydrochloride 36–50 % + isopropanol 20–50 %
Warning
Guanidiniumhydrochlorid
36–50 % + Isopropanol
20–50 %
Achtung
RNase A, lyophilized
Danger
RNase A, lyophilisiert
Gefahr
Hazard phrases
H 226
Flammable liquid and vapour.
H 302
Harmful if swallowed.
H 317
May cause an allergic skin reaction.
H 319
Causes serious eye irritation.
H 334
May cause allergy or asthma symptoms or breathing difficulties if inhaled.
14
Flüssigkeit und Dampf entzündbar.
Gesundheitsschädlich bei Verschlucken.
Kann allergische Hautreaktionen verursachen.
Verursacht schwere Augenreizung.
Kann bei Einatmen Allergie, asthmaartige Symptome oder Atembeschwerden verursachen.
MACHEREY-NAGEL – 05 / 2014, Rev. 03
Genomic DNA from plant
Precaution phrases
P 210
Keep away from heat, hot surfaces, sparks, open flames and other ignition
sources. No smoking.
Von Hitze, heißen Oberflächen, Funken, offenen Flammen sowie anderen
Zündquellenarten fernhalten. Nicht rauchen.
P 233
Keep container tightly closed.
P 261
Avoid breathing dust.
P 280
Wear protective gloves / eye protection.
P 301+312
IF SWALLOWED: Call a POISON CENTER/ doctor/…/if you feel unwell.
P 302+352
IF ON SKIN: Wash with plenty of water/…
P 304+340
IF INHALED: Remove victim to fresh air and keep at rest in a position comfortable for breathing.
Behälter dicht verschlossen halten.
Einatmen von Staub vermeiden.
Schutzhandschuhe / Augenschutz tragen.
BEI VERSCHLUCKEN: Bei Unwohlsein GIFTINFORMATIONSZENTRUM / Arzt /…
anrufen.
BEI KONTAKT MIT DER HAUT: Mit viel Wasser/… waschen.
BEI EINATMEN: An die frische Luft bringen und in einer Position ruhigstellen, die das
Atmen erleichtert.
P 305+351+338 IF IN EYES: Rinse continuously with water for several minutes. Remove
contact lenses if present and easy to do – continue rinsing.
Bei Kontakt mit den Augen: Einige Minuten lang behutsam mit Wasser spülen.
Vorhandene Kontaktlinsen nach Möglichkeit entfernen. Weiter spülen.
P 330
Rinse mouth.
P 333+313
If skin irritation occurs: Get medical advice / attention.
P 337+313
Get medical advice / attention.
P 342+311
If experiencing respiratory symptoms: Call a POISON CENTER/ doctor/…
P 403+235
Store in a well ventilated place. Keep cool.
Mund ausspülen.
Bei Hautreizung: Ärztlichen Rat einholen / ärztliche Hilfe hinzuziehen.
Bei anhaltender Augenreizung: Ärztlichen Rat einholen / ärztliche Hilfe hinzuziehen.
Bei Symptomen der Atemwege: GIFTINFORMATIONSZENTRUM /Arzt/… anrufen.
Kühl an einem gut belüfteten Ort aufbewahren.
For further information please see Material Safety Data Sheets (www.mn-net.com).
Weiterführende Informationen finden Sie in den Sicherheitsdatenblättern (www.mn-net.com).
MACHEREY-NAGEL – 05 / 2014, Rev. 03
15
NucleoSpin® 96 Plant II – centrifuge processing
5
Protocols
5.1 NucleoSpin® 96 Plant II – centrifuge processing
•
For hardware requirements, refer to section 2.3.
•
For use of the NucleoSpin® 96 Plant II Core Kit (REF 740468.4), refer to section
2.4 regarding recommended accessories.
•
For detailed information on each step, see page 18.
Before starting the preparation:
•
Check if Buffer PW2 and RNase A were prepared according to section 3.
•
Equilibrate Buffer PE to 70 °C.
•
Set incubator or oven to 65 °C.
Protocol-at-a-glance
1
Homogenize samples
Up to 100 mg wet or 20 mg
lyophilized plant tissue
5,600–6,000 x g,
2 min
2 a
Cell lysis using Buffer PL1
500 μL PL1
10 μL RNase A
Mix
65 °C, 30 min
Proceed with step 3
2 b
Cell lysis using Buffer PL2
and PL3
400 μL PL2
10 μL RNase A
Mix
65 °C, 30 min
100 μL PL3
Mix and incubate on ice for 5 min
Proceed with step 3
16
MACHEREY-NAGEL – 05 / 2014, Rev. 03
NucleoSpin® 96 Plant II – centrifuge processing
3
Clear lysate by centrifugation
4
Adjust binding conditions
5
Transfer lysate to NucleoSpin®
Plant II Binding Plate
6
Bind DNA to silica membrane
of the NucleoSpin® Plant II
Binding Plate
7
Wash and dry silica membrane
5,600–6,000 x g,
20 min
Mix 450 μL PC with 400 μL cleared lysate
5,600–6,000 x g,
2 min
400 μL PW1
5,600–6,000 x g,
2 min
700 μL PW2
5,600–6,000 x g,
2 min
700 μL PW2
5,600–6,000 x g,
10 min
8
Elute DNA
100 μL PE (70 °C)
(incubate 2 min)
5,600–6,000 x g,
2 min
Repeat once
MACHEREY-NAGEL – 05 / 2014, Rev. 03
17
NucleoSpin® 96 Plant II – centrifuge processing
Detailed protocol
•
•
For hardware requirements, refer to section 2.3.
For use of the NucleoSpin® 96 Plant II Core Kit (REF 740468.4), refer to section
2.4 regarding recommended accessories.
Before starting the preparation:
•
Check if Buffer PW2 and RNase A were prepared according to section 3.
•
Equilibrate Buffer PE to 70 °C.
•
1
Set incubator or oven to 65 °C.
Homogenize samples
Fill up to 100 mg wet plant tissue (or up to 20 mg dried material, for example
lyophilized plant tissue) into each tube of the Tube Strips. Add one 3 mm
diameter steel bead to each tube. Close the tubes with Cap Strips. Freeze
samples in liquid nitrogen. Disrupt cells by vigorous shaking using a mixer mill.
Centrifuge at 5,600 x g for 2 min and remove Cap Strips.
For further processing use either Buffer PL1 (2 a) or Buffers PL2 / PL3 (2 b)!
2 a
Cell lysis using Buffer PL1
Add 500 μL Buffer PL1 and 10 μL RNase A to each sample. Close tubes again
using new Cap Strips (supplied). Mix by vigorous shaking for 15–30 s. Spin
briefly for 30 s at 1,500 x g to collect any sample from the Cap Strips. Incubate
samples at 65 °C for 30 min.
Depending on plant sample and available methods, Buffer PL1 and RNase A
may be added to the plant material before homogenization by the appropriate
mechanical method.
Proceed with step 3.
2 b
Cell lysis using Buffer PL2 and PL3
Add 400 μL Buffer PL2 and 10 μL RNase A to each sample. Close tubes again
using new Cap Strips (supplied). Mix by vigorous shaking for 15–30 s. Spin
briefly for 30 s at 1,500 x g to collect any sample from the Cap Strips. Incubate
samples at 65 °C for 30 min.
Depending on plant sample and available methods, Buffer PL2 and RNase A
may be added to the plant material before homogenization by the appropriate
mechanical method.
Open tubes, add 100 μL Buffer PL3, close tubes, mix thoroughly, and incubate
for 5 min on ice to precipitate SDS completely.
18
MACHEREY-NAGEL – 05 / 2014, Rev. 03
NucleoSpin® 96 Plant II – centrifuge processing
3
Clear lysate by centrifugation
Centrifuge the samples for 20 min at full speed (5,600–6,000 x g). Remove
Cap Strips.
4
Adjust binding conditions
Pre-dispense 450 μL Binding Buffer PC to each well of a MN Square-well
Block. Add 400 μL cleared lysate of each sample and mix by repeated pipetting
up and down. Mix at least 3 times.
5
Transfer lysate to NucleoSpin® Plant II Binding Plate
Place NucleoSpin® Plant II Binding Plate on a MN Square-well Block. Transfer
samples from the previous step into the wells of the NucleoSpin® Plant II
Binding Plate. Do not moisten the rims of the individual wells while dispensing
the samples.
Optional: Seal openings of the binding plate with a Gas-permeable Foil.
6
Bind DNA to silica membrane
Place the NucleoSpin® Plant II Binding Plate stacked on an MN Square-well
Block in the rotor buckets. Centrifuge at 5,600–6,000 x g for 5 min.
Typically, lysates will pass through the columns within 1 min. The centrifugation
process can be extended to 20 min, if the lysates have not passed completely.
7
Wash silica membrane
1st wash
Add 400 μL PW1 to each well of the NucleoSpin® Plant II Binding Plate.
Seal plate with a Gas-permeable Foil and centrifuge again at 5,600–6,000 x g
for 2 min. Place NucleoSpin® Plant II Binding Plate on a new MN Square-well
Block.
2nd wash
Add 700 μL PW2 to each well of the NucleoSpin® Plant II Binding Plate.
Seal plate with a Gas-permeable Foil and centrifuge again at 5,600–6,000 x g
for 2 min.
MACHEREY-NAGEL – 05 / 2014, Rev. 03
19
NucleoSpin® 96 Plant II – centrifuge processing
3rd wash
Add 700 μL PW2 to each well of the NucleoSpin® Plant II Binding Plate.
Seal plate with a Gas-permeable Foil and centrifuge again at 5,600–6,000 x g
for 2 min. Place NucleoSpin® Plant II Binding Plate on a new MN Square-well
Block.
Note: For critical ethanol-sensitive applications, it is recommended to prolong the
centrifugation time up to 15 min or incubate at higher temperature. Remove the
adhesive foil and place the NucleoSpin® Plant II Binding Plate into an incubator for
20 min at 37 °C to evaporate residual ethanol.
8
Elute DNA
Place NucleoSpin® Plant II Binding Plate on the Rack of Tube Strips. Dispense
100 μL pre-heated Buffer PE (70 °C) to each well of the NucleoSpin® Plant II
Binding Plate. Dispense the buffer directly onto the membrane.
Optional: Incubate for 2 min at 70 °C before centrifugation.
Centrifuge at 5,600–6,000 x g for 2 min. Remove the NucleoSpin® Plant II
Binding Plate from the Rack of Tube Strips.
For optimal yield it is recommended to repeat this step once (incubation of
Buffer PE on the membrane not required).
Yields will be 10–20 % higher when eluting with 2 x 100 μL Buffer PE depending
on the total amount of DNA. However, the concentration of DNA will be much
lower than with 100 μL.
Note: Elution can be done with TE buffer (at least pH 8.0) as well. Elution efficiency
will decrease when using elution buffers with pH ≤ 8.0.
20
MACHEREY-NAGEL – 05 / 2014, Rev. 03
NucleoSpin® 96 Plant II – vacuum processing
5.2 NucleoSpin® 96 Plant II – vacuum processing
•
For hardware requirements, refer to section 2.3.
•
For detailed information on each step, see page 24.
•
•
For detailed information regarding the vacuum manifold setup, see page 23.
For use of the NucleoSpin® 96 Plant II Core Kit (REF 740468.4), refer to section
2.4 regarding recommended accessories.
Before starting the preparation:
•
Check if Buffer PW2 and RNase A were prepared according to section 3.
•
Equilibrate Buffer PE to 70 °C.
•
Set incubator or oven to 65 °C.
Protocol-at-a-glance
1
Homogenize samples
Up to 100 mg wet or 20 mg
lyophilized plant tissue
5,600–6,000 x g,
2 min
2 a
Cell lysis using Buffer PL1
500 μL PL1
10 μL RNase A
Mix
65 °C, 30 min
Proceed with step 3
2 b
Cell lysis using Buffer PL2
and PL3
400 μL PL2
Mix
65 °C, 30 min
100 μL PL3
Mix and incubate on ice for 5 min
Proceed with step 3
3
Clear lysate by centrifugation
5,000–6,000 x g
20 min
* Reduction of atmospheric pressure
MACHEREY-NAGEL – 05 / 2014, Rev. 03
21
NucleoSpin® 96 Plant II – vacuum processing
4
Adjust binding conditions
5
Transfer lysate to NucleoSpin®
Plant II Binding Plate
6
Bind DNA to silica membrane
of the NucleoSpin® Plant II
Binding Plate
7
Wash and dry silica membrane
Mix 450 μL PC with 400 μL cleared lysate
-0.2 to -0.4 bar*
(2 min)
400 μL PW1
700 μL PW2
700 μL PW2
-0.4 bar*
(1 min each step)
Remove MN Wash Plate
Dry silica membrane
(10 min, maximum vacuum)
8
Elute DNA
100 μL PE
(incubate 2 min)
-0.4 bar*
(2 min)
Repeat once
22
MACHEREY-NAGEL – 05 / 2014, Rev. 03
NucleoSpin® 96 Plant II – vacuum processing
Setup of vacuum manifold:
Binding / Washing steps
Elution step
Step 4:
Place the NucleoSpin®
Binding Plate on top of
the manifold lid.
Step 4:
Place the NucleoSpin®
Binding Plate on top of
the manifold lid.
Step 3:
Place the manifold lid on
top of the manifold base.
Step 3:
Place the manifold lid on
top of the manifold base.
Step 2:
Place the MN Wash Plate
in the manifold.
Step 2:
Place the Rack of Tube
Strips in the manifold.
MICR
OT
Step 1:
Insert spacers
‘MTP/MULTI-96 PLATE‘
in the manifold base.
MICR
OT
UB
UB
E RA
CK
E RA
CK
Final setup
Step 1:
Insert spacers
‘MICROTUBE RACK‘
in the manifold base.
Final setup
MICR
OT
MICR
OT
UB
UB
E RA
CK
E RA
CK
MACHEREY-NAGEL – 05 / 2014, Rev. 03
23
NucleoSpin® 96 Plant II – vacuum processing
Detailed protocol
•
For hardware requirements, refer to section 2.3.
•
For use of the NucleoSpin® 96 Plant II Core Kit (REF 740468.4), refer to section
2.4 regarding recommended accessories.
•
For detailed information regarding the vacuum manifold setup, see page 23.
Before starting the preparation:
•
Check if Buffer PW2 and RNase A were prepared according to section 3.
•
Equilibrate Buffer PE to 70 °C.
•
•
1
Set incubator or oven to 65 °C.
For detailed information regarding the vacuum manifold set-up see page 23.
Homogenize samples
Fill up to 100 mg wet plant tissue (or up to 20 mg dried, for example
lyophilized, plant tissue) into each tube of the Tube Strips. Add one 3 mm
diameter steel bead to each tube. Close the tubes with Cap Strips. Freeze
samples in liquid nitrogen. Disrupt cells by vigorous shaking using a mixer mill.
Spin at 5,600 x g for 2 min and remove Cap Strips.
For further processing use either Buffer PL1 (2 a) or Buffers PL2 / PL3 (2 b)!
2 a
Cell lysis using Buffer PL1
Add 500 μL Buffer PL1 and 10 μL RNase A to each sample. Close tubes again
using new Cap Strips (supplied). Mix by vigorous shaking for 15–30 s. Spin
briefly for 30 s at 1,500 x g to collect any sample from the Cap Strips. Incubate
samples at 65 °C for 30 min.
Depending on plant sample and available methods, Buffer PL1 and RNase A
may be added to the plant material before homogenization by the appropriate
mechanical method.
Proceed with step 3.
2 b
Cell lysis using Buffer PL2 and PL3
Add 400 μL Buffer PL2 and 10 μL RNase A to each sample. Close tubes again
using new Cap Strips (supplied). Mix by vigorous shaking for 15–30 s. Spin
briefly for 30 s at 1,500 x g to collect any sample from the Cap Strips. Incubate
samples at 65 °C for 30 min.
* Reduction of atmospheric pressure
24
MACHEREY-NAGEL – 05 / 2014, Rev. 03
NucleoSpin® 96 Plant II – vacuum processing
Depending on plant sample and available methods, Buffer PL2 and RNase A
may be added to the plant material before homogenization by the appropriate
mechanical method.
Open tubes, add 100 μL Buffer PL3, close tubes, mix thoroughly, and incubate
for 5 min on ice to precipitate SDS completely.
3
Clear lysate by centrifugation
Centrifuge the samples for 20 min at full speed (5,600–6,000 x g). Remove
Cap Strips.
4
Adjust binding conditions
Pre-dispense 450 μL Binding Buffer PC to each well of a MN Square-well
Block. Add 400 μL cleared lysate of each sample and mix by repeated pipetting
up and down. Mix at least 3 times.
5
Transfer lysate to NucleoSpin® Plant II Binding Plate
Place waste tray into manifold base. Insert spacers labeled ‘MTP/MULTI-96
PLATE’ notched side up into NucleoVac and place the MN Wash Plate on
them. Close manifold and place NucleoSpin® Plant II Binding Plate on top of
the manifold.
Transfer samples from the previous step into the wells of the NucleoSpin®
Plant II Binding Plate. Do not moisten the rims of the individual wells while
dispensing the samples.
6
Bind DNA to silica membrane
Apply vacuum of -0.2 to -0.4 bar* to allow samples to pass through the
membrane. Flow-through rate should be about 1–2 drops per second. Adjust
vacuum strength accordingly. Finally, release the vacuum.
* Reduction of atmospheric pressure
MACHEREY-NAGEL – 05 / 2014, Rev. 03
25
NucleoSpin® 96 Plant II – vacuum processing
7
Wash silica membrane
1st wash
Add 400 μL PW1 to each well of the NucleoSpin® Plant II Binding Plate and
apply vacuum of -0.2 to -0.4 bar* until the buffer has passed the membrane
completely. Release the vacuum.
2nd wash
Add 700 μL PW2 to each well of the NucleoSpin® Plant II Binding Plate and
apply vacuum of -0.2 to -0.4 bar* until the buffer has passed the membrane
completely. Release the vacuum.
3rd wash
Add 700 μL PW2 to each well of the NucleoSpin® Plant II Binding Plate and
apply vacuum of -0.2 to -0.4 bar* until the buffer has passed the membrane
completely. Release the vacuum.
Remove MN Wash Plate and waste tray.
Reassemble the vacuum manifold and dry the membrane by applying maximum
vacuum (-0.6 bar*) for 10 minutes.
8
Elute DNA
Insert spacers ‘MICROTUBE RACK’ into the vacuum manifold base. Place the
Rack of Tube Strips into the manifold base. Close the manifold and insert the
NucleoSpin® Plant II Binding Plate into the manifold top. Dispense 100 μL preheated Buffer PE (70 °C) to each well of the NucleoSpin® Plant II Binding Plate.
Dispense the buffer directly onto the membrane. Incubate at room temperature
for 2 min. Apply vacuum of -0.4 bar* until the elution buffer has passed the
membrane completely.
For optimal yield it is recommended to repeat this step once (incubation of
Buffer PE on the membrane not required)
Yields will be 10–20 % higher when eluting with 2 x 100 μL Buffer PE depending
on the total amount of DNA. However, the concentration of DNA will be much
lower than with 100 μL.
Note: Elution can be done with TE buffer (at least pH 8.0) as well. Elution efficiency
will decrease when using elution buffers with pH ≤ 8.0.
26
MACHEREY-NAGEL – 05 / 2014, Rev. 03
Genomic DNA from plant
6
Appendix
6.1 Troubleshooting
Problem
Possible cause and suggestions
Homogenization of plant material was not sufficient
•
For most species we recommend grinding with steel beads.
Homogenization should be done thoroughly until the plant
material is ground to a fine powder. In most cases this can
be achieved by vigorous shaking for 3 x 60 s with occasional
freezing in liquid nitrogen.
•
This problem can also be avoided by lyophilizing the material.
This way, it will be easier to grind the material.
Extraction of DNA from plant material during lysis was not sufficient
•
To obtain higher yields of DNA, the incubation time in lysis buffer
can be prolonged (up to overnight).
Suboptimal lysis buffer was used
DNA yield is
low
•
Lysis efficiencies of Buffer PL1 (CTAB) and Buffer PL2 (SDS)
are different and depend on the plant species. Try both buffers
in a side-by side purification to find the best detergent system to
lyse your plant material.
Sample contains too much RNA
•
Add 10 μL of RNase A solution to the Lysis Buffer PL1 or PL2
before heat incubation. If this is not successful, add the enzyme
to the cleared supernatant of step 3 and incubate for 30 min at
60 °C.
Sub-optimal Elution
DNA is
degraded
•
The DNA can be either eluted in higher volumes (up to 300 μL)
or by repeating the elution step up to three times. Remember
that the elution buffer must be preheated to 70 °C prior to elution.
•
Also check the pH of the elution buffer used, which should be in
a range of pH 8–8.5. To ensure correct pH, use supplied elution
Buffer PE.
Sample was contaminated with DNase
•
Check bench, pipettes and storage of sample in order to avoid
DNase contamination.
MACHEREY-NAGEL – 05 / 2014, Rev. 03
27
Genomic DNA from plant
Problem
Possible cause and suggestions
Sample contains DNA-degrading contaminants (e.g., phenolic compounds, secondary metabolites)
DNA purity
is low
•
Repeat washing step with Buffer PW1.
Elution buffer contains EDTA
•
EDTA can disturb subsequent reactions. Use of water or
supplied Elution Buffer PE is highly recommended.
6.2 Ordering information
Product
REF
Pack of
NucleoSpin® 96 Plant II
740663 .2
740663 .4
740663 .24
1 x 96 preps
4 x 96 preps
24 x 96 preps
NucleoSpin® 96 Plant II Core Kit
740468 .4
4 x 96 preps
NucleoSpin® 8 Plant II
740669
740669 .5
12 x 8 preps
60 x 8 preps
NucleoSpin® 8 Plant II Core Kit
740467 .4
48 x 8 preps
Buffer PL1
740918
125 mL
Buffer Set PL2 / PL3
740919
1 set
Buffer PC
740937
125 mL
Buffer PW1
740938
125 mL
Buffer PW2 Concentrate
740939
50 mL
RNase A (lyophilized)
740505
740505 .50
100 mg
50 mg
Proteinase K
740506
100 mg
(100 mL Buffer PL2 + 25 mL Buffer PL3)
(for 250 mL Buffer PW2)
28
MACHEREY-NAGEL – 05 / 2014, Rev. 03
Genomic DNA from plant
Product
REF
Pack of
MN Square-well Block
740476
740476 .24
4
24
MN Wash Plate
740479
740479 .24
4
24
Rack of Tube Strips
740477
740477 .24
4 sets
24 sets
Cap Strips
740478
740478 .24
Gas-permeable Foil
740675
50
Self-adhering Foil
740676
50
NucleoVac 96 Vacuum Manifold
740681
1
NucleoVac Vacuum Regulator
740641
1
(1 set consists of 1 rack, 12 strips with 8
tubes each, and 12 Cap Strips)
48
288
Visit www.mn-net.com for more detailed product information.
MACHEREY-NAGEL – 05 / 2014, Rev. 03
29
Genomic DNA from plant
6.3 Product use restriction / warranty
NucleoSpin® 96 Plant II (Core Kit) components are intended, developed, designed,
and sold FOR RESEARCH PURPOSES ONLY, except, however, any other function of
the product being expressly described in original MACHEREY-NAGEL product leaflets.
MACHEREY-NAGEL products are intended for GENERAL LABORATORY USE
ONLY! MACHEREY-NAGEL products are suited for QUALIFIED PERSONNEL ONLY!
MACHEREY-NAGEL products shall in any event only be used wearing adequate
PROTECTIVE CLOTHING. For detailed information please refer to the respective
Material Safety Data Sheet of the product! MACHEREY-NAGEL products shall
exclusively be used in an ADEQUATE TEST ENVIRONMENT. MACHEREY-NAGEL
does not assume any responsibility for damages due to improper application of our
products in other fields of application. Application on the human body is STRICTLY
FORBIDDEN. The respective user is liable for any and all damages resulting from such
application.
DNA/RNA/PROTEIN purification products of MACHEREY-NAGEL are suitable for IN
VITRO-USES ONLY!
ONLY MACHEREY-NAGEL products specially labeled as IVD are also suitable for IN
VITRO-diagnostic use. Please pay attention to the package of the product. IN VITROdiagnostic products are expressly marked as IVD on the packaging.
IF THERE IS NO IVD SIGN, THE PRODUCT SHALL NOT BE SUITABLE FOR IN
VITRO-DIAGNOSTIC USE!
ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY
CLINICAL USE (INCLUDING, BUT NOT LIMITED TO DIAGNOSTIC, THERAPEUTIC
AND/OR PROGNOSTIC USE).
No claim or representations is intended for its use to identify any specific organism
or for clinical use (included, but not limited to diagnostic, prognostic, therapeutic, or
blood banking). It is rather in the responsibility of the user or - in any case of resale of
the products - in the responsibility of the reseller to inspect and assure the use of the
DNA/RNA/protein purification products of MACHEREY-NAGEL for a well-defined and
specific application.
MACHEREY-NAGEL shall only be responsible for the product specifications and the
performance range of MN products according to the specifications of in-house quality
control, product documentation and marketing material.
This MACHEREY-NAGEL product is shipped with documentation stating specifications
and other technical information. MACHEREY-NAGEL warrants to meet the stated
specifications. MACHEREY-NAGEL´s sole obligation and the customer´s sole remedy
is limited to replacement of products free of charge in the event products fail to perform
as warranted. Supplementary reference is made to the general business terms and
conditions of MACHEREY-NAGEL, which are printed on the price list. Please contact
us if you wish to get an extra copy.
There is no warranty for and MACHEREY-NAGEL is not liable for damages or defects
arising in shipping and handling (transport insurance for customers excluded), or
out of accident or improper or abnormal use of this product; defects in products or
30
MACHEREY-NAGEL – 05 / 2014, Rev. 03
Genomic DNA from plant
components not manufactured by MACHEREY-NAGEL, or damages resulting from
such non-MACHEREY-NAGEL components or products.
MACHEREY-NAGEL makes no other warranty of any kind whatsoever, and
SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF
ANY KIND OR NATURE WHATSOEVER, DIRECTLY OR INDIRECTLY, EXPRESS
OR IMPLIED, INCLUDING, WITHOUT LIMITATION, AS TO THE SUITABILITY,
REPRODUCTIVITY, DURABILITY, FITNESS FOR A PARTICULAR PURPOSE OR
USE, MERCHANTABILITY, CONDITION, OR ANY OTHER MATTER WITH RESPECT
TO MACHEREY-NAGEL PRODUCTS.
In no event shall MACHEREY-NAGEL be liable for claims for any other damages,
whether direct, indirect, incidental, compensatory, foreseeable, consequential, or
special (including but not limited to loss of use, revenue or profit), whether based upon
warranty, contract, tort (including negligence) or strict liability arising in connection with
the sale or the failure of MACHEREY-NAGEL products to perform in accordance with
the stated specifications. This warranty is exclusive and MACHEREY-NAGEL makes
no other warranty expressed or implied.
The warranty provided herein and the data, specifications and descriptions of this
MACHEREY-NAGEL product appearing in MACHEREY-NAGEL published catalogues
and product literature are MACHEREY-NAGEL´s sole representations concerning
the product and warranty. No other statements or representations, written or oral, by
MACHEREY-NAGEL´s employees, agent or representatives, except written statements
signed by a duly authorized officer of MACHEREY-NAGEL are authorized; they should
not be relied upon by the customer and are not a part of the contract of sale or of this
warranty.
Product claims are subject to change. Therefore please contact our Technical Service
Team for the most up-to-date information on MACHEREY-NAGEL products. You
may also contact your local distributor for general scientific information. Applications
mentioned in MACHEREY-NAGEL literature are provided for informational purposes
only. MACHEREY-NAGEL does not warrant that all applications have been tested in
MACHEREY-NAGEL laboratories using MACHEREY-NAGEL products. MACHEREYNAGEL does not warrant the correctness of any of those applications.
Last updated: 07 / 2010, Rev. 03
Please contact:
MACHEREY-NAGEL GmbH & Co. KG
Tel.: +49 (0) 24 21 969 270
e-mail: [email protected]
Trademarks / disclaimer:
NucleoSpin® is a registered trademark of MACHEREY-NAGEL GmbH & Co. KG
All used names and denotations can be brands, trademarks, or registered labels of their respective
owner – also if they are not special denotation. To mention products and brands is only a kind of
information (i.e., it does not offend against trademarks and brands and can not be seen as a kind
of recommendation or assessment). Regarding these products or services we can not grant any
guarantees regarding selection, efficiency, or operation.
MACHEREY-NAGEL – 05 / 2014, Rev. 03
31